Analyzes of Molecular Mechanism of Protein Secretion Using Abnormal Protein C as a Model Protein

以异常蛋白C为模型蛋白分析蛋白分泌的分子机制

• 批准号: 05454624
• 负责人: KOIDE Takehiko
• 金额: $ 4.1万
• 依托单位: Himeji Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454624/
• 关键词: Thrombosis; Protein C; Protein C deficiency; Recombinant Mutants; Vitamin K; Warfarin; Endoplasmic reticulum-associated protein degradation; Quality control; 小胞体内分解; プロテインC欠乏症; 異常プロテインC; 遺伝子発現; 蛋白質の膜透過; 蛋白質の細胞内移行

1. ANALYSIS OF SECRETION OF FIVE RECOMBINANT ARG 15 MUTANTS OF PROTEIN C :Protein C (PC) deficiency is classified into two types. Type I shows equivalent reductions of both enzymatic activity and antigen concentration and type II shows only reduction of functional activity. More than 40 mutations have been identified in all over the PC molecule and most of them are caused by a single amino acid replacement through a single base exchange. Among these mutations, the replacement of Arg 15 to Gly in the Gla-domain is known as type II (PC-Yonago) , while the Arg-15 to Trp replacement was reported as either type I or II,and the Arg-15 to Gln mutation as type I.These indicate that amino acid mutations at the 15th position affect both the secretion and function of PC.To elucidate this hypothesis, we mutated Arg-15 to one base changeable amino acids, i.e.G,W,Q,L,or P by the PCR cassette mutation method and constructed pcD2-SRa-PC expression vectors. These vectors were transfected transiently CO … More S-7 cells (2x10^5 cells) by calcium phosphate coprecipitation. The recombinant PC antigen amounts of intracellular and secreted fractions were determined by ELISA (4experiments each) . The amounts of wild type rPC in the intracellular and secreted fractions were 8.4 and 131.3 ng/dish/48h, respectively. The relative amounts of R15G mutant were determined as 133.3? B130.8% (intracellular) and 67.1? B15.9% (secreted) of those of wild type. This suggests that R15G mutant was expressed and secreted nearly equal to normal, which agrees with type II deficiency of the manifestation of this case. The relative amounts of R15W mutant were 55.9? B16.1% (intracellular) and 0.4? B10.1% (secreted) , which strongly suggests that this mutation causes type I deficiency. The R15Q mutant showed 92.2? B16.2% (intracellular) and 75.4? B113.3% (secreted) . Unlike type I manifestation of R15Q,the recombinant was secretory. The R15L and R15P mutants were also secreted by 54.1 and 55.0%, respectively, of that of wild type in the medium.Thus, these two unrecognized mutations may be predicted to cause type II deficiency. Other kidney cell lines derived from human (293 cells) and hamster (BHK-21/C13 cells) also showed similar tendency with that of COS-7 cells.ANALYSIS OF SECRETION OF WARFERIN-TREATED PROTEIN C :Warfarin, an antagonist of vitamin K,is known to disrupt a microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C as well as some other vitamin K-dependent coagulation factors. We studied the effect of warfarin on secretion of recombinant protein C expressed in 293 or BHK cells, or on the endogenous secretion from HepG2 cells. Warfarin caused a two-to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using three cell lines showed that, although protein C was fully secreted in the presence of vitamin K,the decrease in the total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER) -Golgi transport inhibitor (brefeldin A) nor lysosonotropic inhibitors, suggesting that the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C precursor synthesized in the presence of warefarin and the precursor accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion from vitamin K-treated cells. Thus, a cysteine protease (s) appeared to be responsible for the degradation. Inhibitors for intracellular glucosidase I and II,or for mannosidase showed no effect on the degradation. Protein C synthesized in the presence of warfarin was located in the same organelle with protein disulfide isomerase, an ER-resident protein, and the intracellular protein C was sensitive to endoglycosidase H digestion. Moreover, cross-link experiments using DPS suggested that intracellular protein C precursor associated with 100kDa, 80kDa, 40kDa, and 35kDa proteins in the warfarin-treated cells. From these results we conclude that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease (s) in the ER,through a "quality control" mechanism. Less

1. 5个重组ARG - 15蛋白C突变体的分泌分析：蛋白C （PC）缺乏可分为两种类型。I型显示酶活性和抗原浓度的等量降低，II型仅显示功能活性的降低。在整个PC分子中已经发现了40多种突变，其中大多数是由单个碱基交换引起的单个氨基酸替换引起的。在这些突变中，在gla结构域中，Arg-15向Gly的替换被称为II型（PC- yonago），而Arg-15向Trp的替换被报道为I型或II型，Arg-15向Gln的突变被报道为I型。这些表明，第15位的氨基酸突变影响了PC的分泌和功能。为了验证这一假设，我们采用PCR盒式突变方法将Arg-15突变为1个碱基可变氨基酸，即W、Q、L或P，并构建了pcD2-SRa-PC表达载体。用磷酸钙共沉淀法瞬时转染CO…More S-7细胞（2x10^5细胞）。采用ELISA法测定细胞内和分泌部分重组PC抗原的含量（各4个实验）。细胞内和分泌部分野生型rPC含量分别为8.4和131.3 ng/皿/48h。R15G突变体的相对数量为133.3？B130.8%（胞内）和67.1？野生型占15.9%（分泌型）。这表明R15G突变体的表达和分泌与正常几乎相等，这与本病例表现的II型缺陷一致。R15W突变体的相对数量为55.9？B16.1%（细胞内）和0.4？B10.1%（分泌），这强烈表明这种突变导致I型缺陷。R15Q突变体为92.2？B16.2%（细胞内）和75.4？B113.3%（分泌）。与R15Q的I型表现不同，重组蛋白是分泌性的。R15L和R15P突变体在培养基中的分泌量分别为野生型的54.1%和55.0%。因此，这两种未被识别的突变可能会导致II型缺陷。其他来源于人（293细胞）和仓鼠（BHK-21/C13细胞）的肾细胞系也表现出与COS-7细胞相似的趋势。华法林治疗蛋白C的分泌分析：华法林是维生素K的拮抗剂，已知可破坏微粒体维生素K周期，导致血浆蛋白C水平降低，以及一些其他依赖维生素K的凝血因子。我们研究了华法林对293或BHK细胞中表达的重组蛋白C分泌的影响，以及对HepG2细胞内源性分泌的影响。与维生素k处理的细胞相比，华法林导致蛋白质C分泌量减少了2到4倍。使用三个细胞系进行的脉冲追踪实验表明，尽管蛋白C在维生素K存在的情况下完全分泌，但华法林处理的细胞中放射性总量的减少表明细胞内降解。这种降解取决于华法林的浓度，并且不受内质网(ER) -高尔基转运抑制剂（brefeldin A）和溶酶促声抑制剂的抑制，这表明降解发生在高尔基前非溶酶体室中。在所测试的蛋白酶抑制剂中，n -乙酰- leu - leu -methioninal和n -乙酰- leu - leu -norleucinal阻断了在华法林存在下合成的蛋白C前体和细胞内积累的前体的降解，并呈剂量依赖性。然而，这两种抑制剂都不会干扰维生素k处理过的细胞的分泌。因此，半胱氨酸蛋白酶(s)似乎是负责的降解。细胞内葡萄糖苷酶I和II的抑制剂或甘露糖苷酶的抑制剂对降解没有影响。在华法林存在下合成的蛋白C与蛋白二硫异构酶（ER-resident Protein二硫异构酶）位于同一细胞器内，胞内蛋白C对内糖苷酶H的消化非常敏感。此外，DPS交联实验表明，在华法林处理的细胞中，细胞内蛋白C前体与100kDa、80kDa、40kDa和35kDa蛋白相关。从这些结果我们得出结论，在华法林存在下合成的蛋白C通过一种“质量控制”机制被内质网中的半胱氨酸蛋白酶选择性地降解。少

项目成果

Tokunaga,F.,: "Endoplasmic reticulum‐associated degradation of protein C precursor synthesized in the presence of an anticoagulant warfarin.in Blood Coagulation,Fibrinolysis and Platelet." Spring‐Verlag(in press), (1995)

Tokunaga, F.，：“在抗凝剂华法林存在下合成的蛋白 C 前体的内质网相关降解。《血液凝固、纤维蛋白溶解和血小板》（正在出版），(1995 年)”

Tokunaga, F.: "Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum" Biochemistry. 34 (4). 1163-1170 (1995)

Tokunaga, F.：“华法林导致内质网中蛋白 C 前体的降解”生物化学。

徳永 文稔 ほか: "哺乳類細胞を用いた組換え体の発現によるプロテインC異常・欠乏症の分子機構解析" 生化学. 65. 922- (1993)

Fumitoshi Tokunaga 等人：“通过在哺乳动物细胞中表达重组蛋白来分析蛋白 C 异常/缺陷的分子机制”，生物化学，65. 922-(1993)。

Tokunaga,F.,: "Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum." Biochemistry. 34. 1163-1170 (1995)

Tokunaga, F.，：“华法林会导致内质网中蛋白 C 前体的降解。”

Tokunaga, F.: "Endoplasmic reticulum-associated degradation of protein C precursor synthesized in the presence of an anticoagulant, warfarin" Blood Coagulation, Fibrinolysis and Platelet (ed by E.Kakishita) Springer-Verlag. (in press). (1995)

Tokunaga, F.：“在抗凝剂华法林存在下合成的蛋白 C 前体的内质网相关降解”《血液凝固、纤维蛋白溶解和血小板》（由 E.Kakishita 编辑）Springer-Verlag。